Proteases are indispensable constituents of all forms of life including bacteria, and are of major importance in the food, leather, detergent, pharmaceutical, and waste management industries, and in the diagnosis of illness. The amount of proteases used constitutes two-thirds of the total amount of enzymes used in various industries, which is expected to increase.
In addition, a number of proteases involved in blood homeostasis have been purified and characterized from various sources. Some of these proteases are fibrinolytic enzymes that are capable of digesting fibrin. At present, the fibrinolytic agents available for clinical use are mostly plasminogen activators such as a tissue-type plasminogen activator, a urokinase-type plasminogen activator, and a bacterial plasminogen activator streptokinase.
Fibrinolytic enzymes have been purified from fermented food, earthworms (Nakajima N. et al., Biosci. Biotechnol. Biochem. Vol. 57, pp 1726-1730, 1993), and mushrooms (Kim J. H. et al., Biosci. Biotechnol. Biochem. Vol. 65, pp 356-362, 2001) as well as snake venom (Leonardi A. et al., Toxicon. Vol. 40, pp 55-62, 2002). These enzymes, which consist of both serine proteases and metalloproteases, have been suggested as potential sources of oral fibrinolytic drugs. Recently, fibrinolytic enzymes in shark cartilage extract have been characterized. These fibrinolytic activities have correlated with the presence of two proteases in the extract, which were inhibited by 1,10-phenantroline, indicating that the enzymes were metalloproteases (Ratel D. et al., Thromb. Res. Vol. 1115, pp 143-152, 2005).
sPA, uPA, tPA, APSAC, and the like, which are the fibrinolytic agents used for clinical use, can act as a plasminogen activator being capable of producing plasmin to digest fibrin. Such agents disadvantageously show a low specificity to the fibrin, and cause undesired side effects. For example, sPA causes pyrexia, low blood pressure, and allergies. uPA causes bleeding and takes a long time to inject into subjects.
Consequently, the search continues for other fibrinolytic enzymes from various sources for use in thrombolytic therapy.
Screenings for novel enzymes, including proteases, have mainly used the cultivation-dependent approach. Many valuable enzymes originated from cultivable microorganisms; however, the rate of screening for novel enzymes is significantly decreased when standard cultivation methods are used owing to a high rediscovery frequency (Strohl W. R. et al., Drug Discov. Today, Vol. 5, pp 39-41, 2000). In order to use complex communities, efforts to overcome the problem of non-cultivability have been continuously made.